Lecture on Sucrose Density Gradient High-Speed Centrifugation

Introduction

• Topic: Sucrose density gradient high-speed centrifugation
• Purpose: Technique for separating a mixture containing different proteins based on their density.

Background

• Previous Step:
  • Homogenization of starting material (e.g., flies) to isolate proteins, resulting in a mix containing proteins, lipids, carbohydrates.
  • Initial centrifugation to create a protein pellet.
• Current Goal: Separate proteins in the protein pellet.

Sucrose Density Gradient Column

• Purpose: Separate proteins by density.
• Structure:
  • Top: Low-density sucrose.
  • Bottom: High-density sucrose.
  • Middle: Intermediate densities gradually increasing from top to bottom.
• Sucrose: Combination of fructose and glucose.

Process of Separation

1. Resuspension: Protein pellet is resuspended in water.
2. Placement: Resuspended protein is placed at the top of the sucrose density gradient column.
3. Centrifugation:
   • The column is placed in a centrifuge.
   • Proteins move to the sucrose layer matching their density.
   • Separation occurs due to varying densities of proteins:
     • Least dense proteins stay higher in the column.
     • Most dense proteins move lower into denser sucrose layers.

Example

• Proteins: Green, Orange, Pink
  • Green Protein: Least dense
  • Orange Protein: Intermediate density
  • Pink Protein: Most dense
• Outcome: Each protein type settles at the corresponding density layer within the column.

Extraction

• Method: Extract specific layers to isolate proteins of interest.
  • Easier to separate specific proteins from sucrose than from a mixture of different proteins.

Creating a Sucrose Density Gradient

• Steps:
  • Place varying densities of sucrose in a column.
  • Centrifuge to arrange by density.
  • High-density sucrose sinks; low-density remains at the top.

Summary

• Purpose of Technique: Organizes proteins based on density within a sucrose gradient.
• Advantage: Allows for easier separation and purification of specific proteins from a mixture.